The use of mass spectrometers, including those which perform tandem mass spectrometry (MS/MS), are known to be useful in determining amino acid sequences. Currently available spectrometers are capable of presenting a wide mass range of ions, MS/MS providing daughter ion analysis of a single parent mass. The parent ion is selected by use of a frequency of applied supplemental voltage and the daughter ions are ejected by scanning. From this it is possible to obtain a reconstructed parent ion spectrum from a large number of successive daughter ion scans. Parent ions may also be excited by pulses of energy at their resonant frequency, the daughter ions may be ejected from the ion trap at their resonant frequency. The available means for determining unique amino acid sequence lacks specificity.
Ion trap mass spectrometers, such as those manufactured by the Finnigan Corporation, employ an electrostatic field in which ions are formed and stored by use of a quadrupole trapping field. In a tandem quadrupole ion trap mass spectrometer, ions are formed and stored in an ion trap and then mass selected by a mass analyzer wherein the ions are dissociated by means of a collision induced dissociation with a gas or surface and the fragment ions are analyzed by means of a mass or energy analyzer. During the operation of a tandem quadrupole ion trap mass spectrometer product ions, or daughter ions, are produced by isolating a precursor ion, or parent ion, which has undergone the collision induced dissociation. The collision induced dissociation can occur by applying an excitation waveform between end cap electrodes. A radio frequency voltage generator can be used to supply an RF voltage between the end caps and a ring electrode which provides the quadrupole field for trapping ions. A supplemental RF generator is coupled to the end caps to resonate trapped ions at their axial resonant frequencies. These excited ions undergo fragmentation upon the colliding with a surface or buffer gas, such as helium, placed within the buffer trap. An end cap is perforated to allow unstable ions in the field of the ion trap to exit and be detected by an electron multiplier. The magnitude and/or frequency of the fundamental RF voltage can be varied for providing mass selection. The goal is to isolate a particular ion.
Commercially available quadrupole ion trap mass spectrometers capable of tandem mass spectrophotometry are capable of isolating a single parent ion by inducing a collision dissociation to produce daughter ions which are resonantly ejected from an ion trap for detection by a mass-selective instability scan. Alternative techniques that should also be noted include the application of simultaneous excitation voltages or waveforms across the end caps.
After any characteristic daughter ions in the trap have been ejected, application of a parent ion resonant excitation waveform, can result in some parent ions that are resonantly ejected resulting in false positive readings during the detection of the characteristic daughter ions.
Sample mass may be analyzed by any combination of ionization and mass spectrometer. The ionization method may include, but is not limited to such methods as electron ionization, chemical ionization, fast atom bombardment, desorption chemical ionization, plasmadeorption, thermospray, atmospheric pressure chemical ionization, MALDI or electrospray ionization (ESI). Principally the ionization method will be MALDI or ESI. Prior art mass spectrometer formats are known to be useful either singly or in various combinations for use in analyzing translation products. These formats may include, but are not limited to, ionization (I) techniques, including but not limited to matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (e.g., IONSPPAY or THERMOSPRAY), or massive cluster impact (MCI); these ion sources can be matched with detection formats including linear or non-linear reflection time-of-flight (TOF), single or multiple quadropole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof (e.g., ion-trap/time-of-flight). For ionization, numerous matrix/wavelength combinations (MALDI) or solvent combinations (ESI) can be employed.
With regard to the mass spectrometer, per se, there are several types of tandem mass spectrometers that could be used to generate the patterns of MS/MS spectra that are the substrate for the program. These mass spectrometers generate both parent ions in the +1 (MALDI and ESI) and the +2, +3, +4 etc charge state (ESI). The program takes the charge state of the parent into consideration as part of the data associated with the parent ion. The types of mass analyzer mass analyzers include the quadropole, octopole, ion trap or QUISTOR, time of flight (TOF) Time of Flight with reflectron, the Fourier transformed ion cyclotron resonance (FTICR also called FTMS), magnetic and electromagnetic sector.
The tandem mass spectrometer mass consist of the homogenous or heterogenous combination of two or more of any of these devices in any order (Hybrid instrument). The fragmentation method may include post source decay of the parent ion or its fragment, or collision-activated or collision-induced decomposition of the parent ions, or fragment ions, or metastable ions with a relatively immobile target such as gas molecules or other parent or fragment ions, or photons or electrons or solid objects. The combinations may preferentially include the quadropole or octopole ion trap, the Quadropole-Quadropole (DC and rf or rf only)-TOF, the ion trap-FTMS, the TOF-TOF, the ion trap-TOF, or the TOF-PSD-TOF. The signal resulting from the instrument may be mathematically transformed or filtered.
In view of this, sequence determination of an unknown protein or peptide from mass spectral data can be difficult due to the voluminous number of possible sequences consistent with the molecular weight of the amino acid. Multiple samples result in readings that are unacceptable for most applications due to offsets in samples, preparations, device manufacture, noise filtration, technician sample handling, and so forth. Further, recent advancements in amino acid synthesis have allowed the generation of millions of peptide and protein sequences.
Thus, what is needed in the art is a method of analyzing the voluminous amounts of data that is being produced by such devices.